Method for inhibiting C-jun expression using JAK-3 inhibitors

ABSTRACT

The invention provides a method for inhibiting c-jun activation in mammalian or avian cells comprising contacting the cells with a substance that inhibits the activity of Janus family kinase 3 (JAK-3). The invention also provides a therapeutic method for preventing or treating a pathological condition in a mammal wherein c-jun activation is implicated and inhibition of its activation is desired comprising administering to a mammal in need of such therapy, an effective amount of a substance that inhibits the activity of JAK-3. Novel compounds that are JAK-3 inhibitors, as well as pharmaceutical compositions comprising the compounds are also provided.

PRIORITY OF INVENTION

[0001] This application claims priority under 35 U.S.C. §19(e) from U.S.Provisional Application No. 60/091,150, filed Jun. 30, 1998.

BACKGROUND OF THE INVENTION

[0002] The protooncogene c-jun is the cellular counterpart of the v-junoncogene of avian sarcoma virus 17. C-jun expression is activated inresponse to a diverse set of DNA-damaging agents including ara-C, UVradiation, topoisomerase II inhibitors, alkylating agents, and ionizingradiation. As an immediate early response gene that is rapidly inducedby pleiotropic signals, c-jun may have important regulatory functionsfor cell cycle progression, proliferation, and survival. See Ryder, K.,Lau, L. F., and Nathans, D. “A gene activated by growth factors isrelated to the oncogene v-jun,” Proc Natl Acad Sci USA. 85: 1487-1491,1988; Schutte, J., Viallet, J., Nau, M., Segal, S., Fedorko, J., andMinna, J. “jun-B inhibits and c-fos stimulates the transforming andtrans-activating activities of c-jun, Cell. 59: 987-997, 1989; Neuberg,M., Adamkiewicz, J., Hunter, J. B., and Mueller, R. “A fos proteincontaining the Jun leucine zipper forms a homodimer which binds to theAP-1 binding site,” Nature. 341: 589-590, 1989; Mitchell, P. J. andTjian, R. “Transcriptional regulation in mammalian cells bysequence-specific DNA binding proteins,” Science. 245: 371-378, 1989;Bohmann, D., Bos, T. J., Admon, T., Nishimura, R., Vogt, P. K., andTijian, R. “Human protooncogene c-jun encodes a DNA binding protein withstructural and functional properties of transcription factor AP-1,”Science. 238: 1386-1392, 1988; Kharbanda, S. M., Sherman, M. L., andKufe, D. W. “Transcriptional regulation of c-jun gene expression byarabinofuranosylcytosine in human myeloid leukemia cells,” J ClinInvest. 86: 1517-1523, 1990; Rosette, C. and Karin, M. “Ultravioletlight and osmotic stress: activation of the JNK cascade through multiplegrowth factor and cytokine receptors,” Science. 274: 1194-7, 1996;Rubin, E., Kharbanda, S., Gunji, H., and Kufe, D. “Activation of thec-jun protooncogene in human myleloid leukemia cells treated withetoposide,” Molecular Pharmacology. 39: 697-701, 1991; Dosch, J. andKaina, B. “Induction of c-fos, c-jun, junB and junD mRNA and AP-1 byalkylating mutagens in cells deficient and proficient for the DNA repairprotein O6-methylguanine-DNA methyltransferase (MGMT) and itsrelationship to cell death, mutation induction and chromosomalinstability,” Oncogene. 13: 1927-35, 1996; Chae, H. P., Jarvis, L. J.,and Uckun, F. M. “Role of tyrosine phosphorylation in radiation-inducedactivation of c-jun protooncogene in human lymphohematopoietic precursorcells,” Cancer Res. 53: 447-51, 1993; and Karin, M., Liu, Z.-G., andZandi, E. “AP-1 function and regulation,” Current Opinion in CellBiology. 9: 240-246, 1997.

[0003] C-jun encodes the nuclear DNA-binding protein, JUN, that containsa leucine-zipper region involved in homo- and heterodimerization. JUNprotein dimerizes with another JUN protein or the product of c-fos geneand forms the activating protein-1 (AP-1) transcription factor. JUN-JUNhomodimers and JUN-FOS heterodimers preferentially bind to a specificheptameric consensus sequence found in the promoter region of multiplegrowth regulatory genes. Alterations of c-jun protooncogene expressioncan therefore modulate the transcription of several growth-regulatorsaffecting cell proliferation and differentiation. See Ryder, K., Lau, L.F., and Nathans, D. “A gene activated by growth factors is related tothe oncogene v-jun,” Proc Natl Acad Sci USA. 85: 1487-1491, 1988;Neuberg, M., Adamkiewicz, J., Hunter, J. B., and Mueller, R. “A fosprotein containing the Jun leucine zipper forms a homodimer which bindsto the AP-1 binding site,” Nature. 341: 589-590, 1989; Karin, M., Liu,Z.-G., and Zandi, E. “AP-1 function and regulation,” Current Opinion inCell Biology. 9: 240-246, 1997; Angel, P., Allegretto, E. A., Okino, S.T., Hattori, K., Boyle, W. J., Hunter, T., and Karin, M. “Oncogene junencodes a sequence-specific trans-activator similar to AP-1,” Nature.332: 166-170, 1988; and Musti, A. M., Treier, M., and Bohmann, D.“Reduced ubiquitin-dependent degradation of c-Jun after phosphorylationby MAP kinases,” Science. 275: 400-402, 1997.

[0004] C-jun plays a pivotal role in Ras-induced transformation and hasalso been implicated as a regulator of apoptosis when de novo proteinsynthesis is required. C-jun induction is required for ceramide-inducedapoptosis and stress-induced apoptosis after UV exposure or other formsof DNA damage. This induction is thought to be triggered by activationof JUN-N-terminal kinases (JNKs) (also known as stress-activated proteinkinases) which leads to enhanced cjun transcription by phosphorylationof JUN at sites that increases its ability to activate transcription.Ectopic expression of a dominant-negative c-jun mutant lacking the Nterminus or a dominant-negative JNK kinase abolishes stress-inducedapoptosis. See Karin, M., Liu, Z.-G., and Zandi, E. “AP-1 function andregulation,” Current Opinion in Cell Biology. 9: 240-246, 1997;Collotta, F., Polentarutti, N., and Mantovani, A. “Expression andinvolvement of c-fos and c-jun protooncogenes in programmed cell deathinduced by growth factor deprivation in lymphoid cell lines,” J. Biol.Chem. 267: 18278-18283, 1992; Ham, J., Babij, C., Whitfield, J., Pfarr,C. M., Lallemand, D., Yaniv, M., and Rubin, L. L. “A c-Jun dominantnegative mutant protects sympathetic neurons against programmed celldeath,” Neuron. 14: 927-939, 1995; Verheij, M., Bose, R., Lin, X. H.,Yao, B., Jarvis, W. D., Grant, S., Birrer, K M. J., Szabo, E., Zon, L.I., Kyriakis, J. M., Haimovitz F A., Fuks, Z., and Kolesnick, R. N.“Requirement for ceramide-initiated SAPK/JNK signalling instress-induced apoptosis,” Nature. 380: 75-9, 1996; Hibi, M., Lin, A.,Smeal, T., Minden, A., and Karin, M. “Identification of an oncoprotein-and UV-responsive protein kinase that binds and potentiates the c-Junactivation domain,” Genes Dev. 7: 2135-48, 1993; Derijard, B., Hibi, M.,Wu, I. H., Barrett, T., Su, B., Deng, T., Karin, M., and Davis, R. J.“JNK1: a protein kinase stimulated by UV light and Ha-Ras that binds andphosphorylates the c-Jun activation domain,” Cell. 76: 1025-37, 1994;and Chen, Y. R., Wang, X., Templeton, D., Davis, R. J., and Tan, T. H.“The role of c-Jun N-terminal kinase (JNK) in apoptosis induced byultraviolet C and gamma radiation. Duration of JNK activation maydetermine cell death and proliferation,” JBiol Chem. 271: 31929-36,1996.

[0005] Protein tyrosine kinases (PTK) play important roles in theinitiation and maintenance of biochemical signal transduction cascadesthat affect proliferation and survival of B-lineage lymphoid cells.Oxidative stress has been shown to activate BTK, SYK, and Src familyPTK. It is known that PTK activation precedes and mandatesradiation-induced activation of c-jun protooncogene expression in humanB-lineage lymphoid cells (Chae, H. P., Jarvis, L. J., and Uckun, F. M.Cancer Res. 53: 447-51, 1993). However, the identity of the PTKresponsible for radiation-induced c-jun activation is not yet known. SeeUckun, F. M., Waddick, K. G., Mahajan, S., Jun, X., Takata, M., Bolen,J., and Kurosaki, T. “BTK as a mediator of radiation-induced apoptosisin DT-40 lymphoma B cells,” Science. 273: 1096-100, 1996; Kurosaki, T.“Molecular mechanisms in B cell antigen receptor signaling,” Curr OpinImmunol. 9: 309-18, 1997; Uckun F. M, Evans W. E, Forsyth C. J, WaddickK. G, T-Ahlgren L., Chelstrom L. M, Burkhardt A., Bolen J., Myers D. E.“Biotherapy of B-cell precursor leukemia by targeting genistein toCD19-associated tyrosine kinases.” Science 267:886-891, 1995; Myers D.E., Jun X., Waddick K. G., Forsyth C., Chelstrom L. M., Gunther R. L.,Turner N. E, Bolen J., Uckun F. M. “Membrane-associated CD19-LYN complexis an endogenous p53-independent and bcl-2-independent regulator ofapoptosis in human B-lineage lymphoma cells. “Proc Nat'l Acad Sci USA92: 9575-9579, 1995; Tuel Ahlgren, L., Jun, X., Waddick, K. G., Jin, J.,Bolen, J., and Uckun, F. M. “Role of tyrosine phosphorylation inradiation-induced cell cycle-arrest of leukemic B-cell precursors at theG2-M transition checkpoint,” Leuk Lymphoma. 20: 417-26, 1996; Qin, S.,Minami, Y., Hibi, M., Kurosaki, T., and Yamamura, H. “Syk-dependentand—independent signaling cascades in B cells elicited by osmotic andoxidative stress,” J Biol Chem. 272: 2098-103, 1997; Saouaf, S. J.,Mahajan, S., Rowley, R. B., Kut, S., Fargnoli, J., Burkhardt, A. L.,Tsukada, S., Witte, O. N., and Bolen, J. B. “Temporal differences in theactivation of three classes of non-transmembrane protein tyrosinekinases following B cell antigen receptor surface engagement,” Proc NatlAcad Sci USA. 91: 9524-28, 1994; Law, D. A., Chan, V. F. W., Datta, S.K., and DeFranco, A. L. “B-cell antigen receptor motifs have redundantsignalling capabilities and bind the tyrosine kinases PTK72,Lyn andFyn,” Curr Biol. 3: 645-57, 1993; Hibbs, M. L., Tarlinton, D. M., Armes,J., Grail, D., Hodgson, G., Maglitto, R., Stacker, S. A., and Dunn, A.R. “Multiple defects in the immune system of Lyn-deficient mice,culminating in autoimmune disease,” Cell. 83: 301-311, 1995; Aoki, Y.,Isselbacker, K. J., and Pilai, S. “Bruton tyrosine kinase is tyrosinephosphorylated and activated in pre-B lymphocytes and receptor-ligated Bcells,” Proc Natl Acad Sci USA. 91: 10606-10609, 1994; Jugloff, L. S.and Jongstra Bilen, J. “Cross-linking of the IgM receptor induces rapidtranslocation of IgM-associated Ig alpha, Lyn, and Syk tyrosine kinasesto the membrane skeleton, J Immunol. 159: 1096-106, 1997; Thomis, D. S.,Gurniak, C. B., Tivol, E., Sharpe, A. H., and Berg, L. J.” Defects in Blymphocyte maturation and T lymphocyte activation in mice lacking Jak3,” Science. 270: 794-797, 1995; Nosaka, T., Van Deursen, J. M., Tripp,R.A., Thierfelder, W. E., Witthuhn, B. A., McMickle, A. P., Doherty, P.c., Grosveld, G. C., and Ihle, J. N. “Defective lymphoid development inmice lacking Jak 3,” Science. 270: 800-802, 1995.

[0006] U.S. patent application Ser. No. 09/087,479 (entitledQuinazolines For Treating Brain Tumor; filed May 28, 1998) discloseshydroxyquinazoline derivatives that exhibit potent cytotoxicity againsthuman glioblastoma cells (i.e. brain tumor cells). Because JAK-3 is notknown to be present in these glioblastoma cells, the cytotoxic activityof the hydroxyquinazoline derivatives is not believed to result frominhibition of JAK-3 activity. Additionally, the cytotoxic activity ofthe hydroxyquinazoline derivatives is not known to result from theinhibition of c-jun activation.

[0007] There is currently a need for therapeutic agents and methods thatare useful for preventing or reducing cell damage that results fromexposure to radiation and chemical agents that cause DNA-damage. Thereis also a need for chemical agents as well as in vitro and in vivomethods that can be used to further investigate the biological pathwaysassociated with DNA-damage that results from exposure to radiation orchemical agents.

SUMMARY OF THE INVENTION

[0008] The invention provides a method comprising inhibiting c-junexpression in cells (e.g. mammalian or avian) by contacting the cells(in vitro or in vivo) with a substance that inhibits the activity ofJanus family kinase 3 (JAK-3).

[0009] The invention also provides a therapeutic method for preventingor treating a pathological condition in a mammal (e.g. a human) whereinc-jun activation is implicated and inhibition of its expression isdesired comprising administering to a mammal in need of such therapy, aneffective amount of a substance that inhibits the activity of JAK-3.

[0010] The invention also provides novel compounds of formula I as wellas processes and intermediates useful for their preparation.

[0011] The invention also provides substances that are effective toinhibit JAK-3 for use in medical therapy (preferably for use in treatingconditions that result from exposure to radiation or to chemical agentsthat cause DNA damage), as well as the use of substances that inhibitJAK-3 for the manufacture of a medicament for the treatment of acondition that is associated with exposure to radiation, or to chemicalagents that cause DNA damage.

BRIEF DESCRIPTION OF THE FIGURES

[0012]FIG. 1. Radiation-induced c-jun activation in wild-type DT40lymphoma B-cells. [A]. Dose response for induction of c-jun mRNA. DT-40chicken cells were irradiated at the indicated doses (0,10,15,20 Gy).Total RNA was extracted after a 2 hours or 4 hours post-irradiation timeperiod. RNA (20 mg) was loaded on a Northern gel and transferred bycapillary blotting to a nylon membrane. The Northern blot was hybridizedwith a ³²P labeled chicken c-jun probe (top panel) or a chicken GAPDHprobe (bottom panel). The inset shows the values for the c-jun/GAPDHtranscript expression ratios as determined with a Bio Rad StoragePhosphor Imager and corresponding SI values [B]. Effect of the PTKinhibitor genistein on induction of c-jun mRNA. Cells were treated with30 mg/ml of genistein for 24 hours at 37° C. prior to exposure to 20 Gyionizing radiation. c-jun expression levels were determined as in [A].

[0013]FIG. 2. Radiation-induced activation of c-jun in BTK-DT-40 cells.Two representative experiments (shown in [A] and [B]) showing inductionof c-jun mRNA expression by ionizing radiation in wild type (WT) andBTK⁻ DT-40 cells. Poly (A)⁺ RNA was isolated from non-irradiated cellsas well as irradiated cells (20 Gy, with a 2 hours post-radiationrecovery period). Northern blots of 2 mg of poly (A)+were hybridizedwith c-jun probe (top panel), (-actin probe (middle panel in [A] only),and GAPDH probe (bottom panel). The inset below each panel shows therelative expression of c-jun normalized for RNA load (c-jun/GAPDH ratio)and SI (fold induction over non-irradiated controls).

[0014]FIG. 3. Induction of c-jun mRNA expression by ionizing radiationin wild type and mutant DT40 cell lines. DT-40, BTK⁻ DT-40, SYK⁻ DT-40(shown in [A]), as well as LYN⁻ DT-40 and LYN SYK DT 40 cells (shown in[B]) were irradiated with 20 Gy and poly (A)⁺ RNA (in [A]) or total RNA(in [B]) was harvested after a 2 hour recovery period. RNA fromnon-irradiated cells was used as a control. Northern blots containing 2mg of poly (A)⁺ (in [A]) or 20 mg of total RNA (in [B]) from each cellline were hybridized with both ³² P labeled c-jun probe(top panel) andGAPDH probe (bottom panel). The insets below the panels show therelative expression of c-jun normalized for RNA loading (c-jun/GAPDHratios) as well as the SI (fold induction over non-irradiated controls).

[0015]FIG. 4. JAK-3 Inhibitors. [A]. Structures of JAK-3 inhibitors. [B]Specificity of JAK-3 inhibitors. Sf21 cells infected with baculovirusexpression vectors for JAK-1 JAK-2 or JAK-3 were subjected toimmunoprecipitation with anti-JAK antibodies. JAK-1 (shown in B.1),JAK-2 (shown in B.2) and JAK-3 (shown in B.3 and B.4 which illustrateresults from 2 independent experiments) immune complexes were treatedwith 1% DMSO (vehicle control =CON), Compound 1, or Compound 2 for 1hour prior to hot kinase assays, as described (20,22). Both compoundsinhibited JAK-3 when used at 10 μg/ml whereas they did not inhibit JAK-1or JAK-2 even at 75 μg/ml [C]. EMSAs of 32Dc22-IL-2Rβ cells. Compound1(100 (g/ml) and Compound 2 (100 (g/ml) inhibited IL-2 triggeredJAK-3-dependent STAT activation but not IL-3-triggeredJAK-1/JAK-2-dependent STAT activation in 32Dc11-IL-2Rβ cells.

[0016]FIG. 5. Effects of a JAK-3 inhibitor on c-jun induction inirradiated DT40 cells. Cells were treated with the quinazolinederivative4-(3′-Bromo-4′-hydroxyl-phenyl)-amino-6,7-dimethoxyquinazoline (100mg/ml) for 24 hours at 37° C. prior to exposure to 20 Gy ionizingradiation. c-jun expression levels were determined as outlined in FIGS.1-3.

DETAILED DESCRIPTION

[0017] As used herein, the term “inhibit” means to reduce by ameasurable amount, or prevent entirely; and the phrase “inhibit c-junactivation” includes the inhibition of RNA production and the inhibitionof the production of the protein encoded by the RNA.

[0018] Applicants examined the potential involvement of BTK, SYK and LYNin radiation-induced c-jun activation, using DT40 chicken lymphomaB-cell clones rendered deficient for these specific PTK by targeted genedisruption. It was found that BTK plays no role in radiation-inducedc-jun activation. Similarly, neither LYN nor SYK are required foractivation of c-jun after radiation exposure. However, theirparticipation may influence the magnitude of the c-jun response. It wasunexpectedly discovered, however, that an inhibitor of Janus familykinase 3 (JAK-3) abrogated radiation-induced c-jun activation.

[0019] C-jun expression can be activated by exposure to chemical agentsthat damage DNA such as ara-C, a topoisomerase II inhibitors, oralkylating agents. C-jun activation can also result from exposure toultraviolet radiation or ionizing radiation. According to the invention,inhibitors of JAK-3 can be used to inhibit c-jun expression resultingfrom exposure to radiation or exposure to chemical agents.

[0020] The methods of the invention can be carried out in vitro. Such invitro methods are also useful for studying the biological processesassociated with cell response to DNA damaging agents. The methods of theinvention can also be carried out in vivo. Such methods can also be usedto study the biological processes associated with cell response to DNAdamaging agents, as well as for treating pathological conditions inmammals (e.g. humans) that result from exposure to DNA-damaging agents.

[0021] Pathological conditions that result from exposure to DNA-damagingagents include conditions that result from oxidative stress, such astissue or organ (e.g. heart, liver, or kidney) damage, inflammation, andhair loss, as well as the negative effects that are produced by oxygenfree radicals during chemotherapy. Oxidative stress may result fromexposure to external agents, or may result from internal processes.Thus, JAK-3 inhibitors are also useful for treating conditions resultingfrom the action of internally generated oxygen free radicals, such asaging and amyelotrophic lateral sclerosis (ALS).

[0022] According to the invention, the JAK-3 inhibitors may beadministered prophylactically, i.e. prior to exposure to theDNA-damaging agent, or the JAK-3 inhibitors may be administered afterexposure to the DNA damaging agent.

[0023] The JAK-3 inhibitors useful in the methods of the inventioninclude all compounds capable of inhibiting the activity of JAK-3, itbeing well known in the art how to measure a compounds ability toinhibit JAK-3, for example, using standard tests similar to the testdescribed hereinbelow in Example 2 under the heading “Effects of a JAK-3inhibitor on radiation-induced c-jun activation in DT40 cells.”

[0024] JAK-3 inhibitors that are useful in the methods of the inventioninclude compounds of formula I:

[0025] wherein

[0026] X is HN, R₁₁N, S, O, CH₂, or R₁₁CH;

[0027] R₁₁ is hydrogen, (C₁-C₄)alkyl, or (C₁-C₄)alkanoyl;

[0028] R₁-R₈ are each independently hydrogen, hydroxy, mercapto, amino,nitro, (C₁-C₄)alkyl, (C₁-C₄)alkoxy, (C₁-C₄)alkylthio, or halo; whereintwo adjacent groups of R₁R₅ together with the phenyl ring to which theyare attached may optionally form a fused ring, for example forming anaphthyl or a tetrahydronaphthyl ring; and further wherein the ringformed by the two adjacent groups of R₁-R₅ may optionally be substitutedby 1, 2, 3, or 4 hydroxy, mercapto, amino, nitro, (C₁-C₄)alkyl,(C₁-C₄)alkoxy, (C₁-C₄)alkylthio, or halo; and

[0029] R₉ and R₁₀ are each independently hydrogen, (C₁-C₄)alkyl,(C₁-C₄)alkoxy, halo, or (C₁-C₄)alkanoyl; or R₉ and R₁₀ together aremethylenedioxy; or a pharmaceutically acceptable salt thereof.

[0030] The following definitions are used, unless otherwise described:halo is fluoro, chloro, bromo, or iodo. Alkyl, alkanoyl, etc. denoteboth straight and branched groups; but reference to an individualradical such as “propyl” embraces only the straight chain radical, abranched chain isomer such as “isopropyl” being specifically referredto. (C₁-C4)Alkyl includes methyl, ethyl, propyl, isopropyl, butyl,iso-butyl, and sec-butyl; (C₁-C₄)alkoxy includes methoxy, ethoxy,propoxy, isopropoxy, butoxy, iso-butoxy, and sec-butoxy; and(C₁-C₄)alkanoyl includes acetyl, propanoyl and butanoyl.

[0031] A specific group of compounds are compounds of formula I whereinR₁-R₅ are each independently hydrogen, mercapto, amino, nitro,(C₁-C₄)alkyl, (C₁-C₄)alkoxy, (C₁-C₄)alkylthio, or halogen.

[0032] Another specific group of compounds are compounds of formula Iwherein R₉ and R₁₀ are each independently hydrogen, (C₁-C₄)alkyl, halo,or (C₁-C₄)alkanoyl; or R₉ and R₁₀ together are methylenedioxy; or apharmaceutically acceptable salt thereof.

[0033] JAK-3 inhibitors that are useful in the methods of the inventionalso include compounds of formula I as described in U.S. patentapplication Ser. No. 09/087,479 (entitled Quinazolines For TreatingBrain Tumor; filed May 28, 1998).

[0034] Preferred JAK-3 inhibitors include4-(4′-hydroxylphenyl)-amino-6,7-dimethoxyquinazoline and4-(3′-bromo-4′-hydroxylphenyl)-arnino-6,7-dimethoxyquinazoline, or apharmaceutically acceptable salt thereof.

[0035] Substances that inhibit JAK-3 (“the Substance(s)”) can beformulated as pharmaceutical compositions and administered to amammalian host, such as a human patient in a variety of forms adapted tothe chosen route of administration, i.e., orally or parenterally, byintravenous, intramuscular, topical or subcutaneous routes.

[0036] Thus, the Substances may be systemically administered, e.g.,orally, in combination with a pharmaceutically acceptable vehicle suchas an inert diluent or an assimilable edible carrier. They may beenclosed in hard or soft shell gelatin capsules, may be compressed intotablets, or may be incorporated directly with the food of the patient'sdiet. For oral therapeutic administration, the Substance may be combinedwith one or more excipients and used in the form of ingestible tablets,buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers,and the like. Such compositions and preparations should contain at least0.1% of the Substance. The percentage of the compositions andpreparations may, of course, be varied and may conveniently be betweenabout 2 to about 60% of the weight of a given unit dosage form. Theamount of Substance in such therapeutically useful compositions is suchthat an effective dosage level will be obtained.

[0037] The tablets, troches, pills, capsules, and the like may alsocontain the following: binders such as gum tragacanth, acacia, cornstarch or gelatin; excipients such as dicalcium phosphate; adisintegrating agent such as corn starch, potato starch, alginic acidand the like; a lubricant such as magnesium stearate; and a sweeteningagent such as sucrose, fructose, lactose or aspartame or a flavoringagent such as peppermint, oil of wintergreen, or cherry flavoring may beadded. When the unit dosage form is a capsule, it may contain, inaddition to materials of the above type, a liquid carrier, such as avegetable oil or a polyethylene glycol. Various other materials may bepresent as coatings or to otherwise modify the physical form of thesolid unit dosage form. For instance, tablets, pills, or capsules may becoated with gelatin, wax, shellac or sugar and the like. A syrup orelixir may contain the active compound, sucrose or fructose as asweetening agent, methyl and propylparabens as preservatives, a dye andflavoring such as cherry or orange flavor. Of course, any material usedin preparing any unit dosage form should be pharmaceutically acceptableand substantially non-toxic in the amounts employed. In addition, theSubstance may be incorporated into sustained-release preparations anddevices.

[0038] The Substances may also be administered intravenously orintraperitoneally by infusion or injection. Solutions of the Substancecan be prepared in water, optionally mixed with a nontoxic surfactant.Dispersions can also be prepared in glycerol, liquid polyethyleneglycols, triacetin, and mixtures thereof and in oils. Under ordinaryconditions of storage and use, these preparations contain a preservativeto prevent the growth of microorganisms.

[0039] The pharmaceutical dosage forms suitable for injection orinfusion can include sterile aqueous solutions or dispersions or sterilepowders comprising the Substance which are adapted for theextemporaneous preparation of sterile injectable or infusible solutionsor dispersions, optionally encapsulated in liposomes. In all cases, theultimate dosage form must be sterile, fluid and stable under theconditions of manufacture and storage. The liquid carrier or vehicle canbe a solvent or liquid dispersion medium comprising, for example, water,ethanol, a polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycols, and the like), vegetable oils, nontoxic glycerylesters, and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the formation of liposomes, by themaintenance of the required particle size in the case of dispersions orby the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, buffers or sodiumchloride. Prolonged absorption of the injectable compositions can bebrought about by the use in the compositions of agents delayingabsorption, for example, aluminum monostearate and gelatin.

[0040] Sterile injectable solutions are prepared by incorporating theSubstance in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfilter sterilization. In the case of sterile powders for the preparationof sterile injectable solutions, the preferred methods of preparationare vacuum drying and the freeze drying techniques, which yield a powderof the active ingredient plus any additional desired ingredient presentin the previously sterile-filtered solutions.

[0041] For topical administration, the Substances may be applied in pureform, i.e., when they are liquids. However, it will generally bedesirable to administer them to the skin as compositions orformulations, in combination with a dermatologically acceptable carrier,which may be a solid or a liquid.

[0042] Useful solid carriers include finely divided solids such as talc,clay, microcrystalline cellulose, silica, alumina and the like. Usefulliquid carriers include water, alcohols or glycols orwater-alcohol/glycol blends, in which the Substances can be dissolved ordispersed at effective levels, optionally with the aid of non-toxicsurfactants. Adjuvants such as fragrances and additional antimicrobialagents can be added to optimize the properties for a given use. Theresultant liquid compositions can be applied from absorbent pads, usedto impregnate bandages and other dressings, or sprayed onto the affectedarea using pump-type or aerosol sprayers.

[0043] Thickeners such as synthetic polymers, fatty acids, fatty acidsalts and esters, fatty alcohols, modified celluloses or modifiedmineral materials can also be employed with liquid carriers to formspreadable pastes, gels, ointments, soaps, and the like, for applicationdirectly to the skin of the user.

[0044] Examples of useful dermatological compositions which can be usedto deliver the Substances to the skin are known to the art; for example,see Jacquet et al. (U.S. Pat. No. 4,608,392), Geria (U.S. Pat. No.4,992,478), Smith et al. (U.S. Pat. No. 4,559,157) and Wortzman (U.S.Pat. No. 4,820,508).

[0045] Useful dosages of the compounds of formula I can be determined bycomparing their in vitro activity, and in vivo activity in animalmodels. Methods for the extrapolation of effective dosages in mice, andother animals, to humans are known to the art; for example, see U.S.Pat. No. 4,938,949.

[0046] Generally, the concentration of the Substance in a liquidcomposition, such as a lotion, will be from about 0.1-25 wt-%,preferably from about 0.5-10 wt-%. The concentration in a semi-solid orsolid composition such as a gel or a powder will be about 0.1-5 wt-%,preferably about 0.5-2.5 wt-%.

[0047] The amount of the Substance required for use in treatment willvary not only with the particular salt selected but also with the routeof administration, the nature of the condition being treated and the ageand condition of the patient and will be ultimately at the discretion ofthe attendant physician or clinician.

[0048] In general, however, a suitable dose will be in the range of fromabout 0.5 to about 100 mg/kg, e.g., from about 10 to about 75 mg/kg ofbody weight per day, such as 3 to about 50 mg per kilogram body weightof the recipient per day, preferably in the range of 6 to 90 mg/kg/day,most preferably in the range of 15 to 60 mg/kg/day.

[0049] The Substance is conveniently administered in unit dosage form;for example, containing 5 to 1000 mg, conveniently 10 to 750 mg, mostconveniently, 50 to 500 mg of active ingredient per unit dosage form.

[0050] Ideally, the Substance should be administered to achieve peakplasma concentrations of from about 0.5 to about 75 μM, preferably,about 1 to 50 μM, most preferably, about 2 to about 30 μM. This may beachieved, for example, by the intravenous injection of a 0.05 to 5%solution of the Substance, optionally in saline, or orally administeredas a bolus containing about 1-100 mg of the Substance. Desirable bloodlevels may be maintained by continuous infusion to provide about0.01-5.0 mg/kg/hr or by intermittent infusions containing about 0.4-15mg/kg of the Substance.

[0051] The Substance may conveniently be presented in a single dose oras divided doses administered at appropriate intervals, for example, astwo, three, four or more sub-doses per day. The sub-dose itself may befurther divided, e.g., into a number of discrete loosely spacedadministrations; such as multiple inhalations from an insufflator or byapplication of a plurality of drops into the eye.

[0052] The invention will now be illustrated by the followingnon-limiting Examples.

EXAMPLES Example 1

[0053] Chemical synthesis and Characterization of JAK-3 Inhibitors

[0054] Melting points are uncorrected. ¹H NMR spectra were recordedusing a Varian Mercury 300 spectrometer in DMSO-d₆ or CDCl₃. Chemicalshifts are reported in parts per million (ppm) with tetramethylsilane(TMS) as an internal standard at zero ppm. Coupling constant (J) aregiven in hertz and the abbreviations s, d, t, q, and m refer to singlet,doublet, triplet, quartet and multiplet, respectively. Infrared spectrawere recorded on a Nicolet PROTEGE 460-IR spectrometer. Massspectroscopy data were recorded on a FINNIGAN MAT 95, VG 7070E-HF G.C.system with an HP 5973 Mass Selection Detector. UV spectra were recordedon BECKMAN DU 7400 and using MeOH as the solvent. TLC was performed on aprecoated silica gel plate (Silica Gel KGF; Whitman Inc). Silica gel(200-400 mesh, Whitman Inc.) was used for all column chromatographyseparations. All chemicals were reagent grade and were purchased fromAldrich Chemical Company (Milwaukee, Wis) or Sigma Chemical Company (St.Louis, Mo.).

[0055] The common synthetic precursor 4-chloro-6,7-dimethoxyquinazoline(7), used for preparing compounds (1) and (2), was prepared usingliturature procedures as illustrated in Scheme 1.

[0056] 4,5-Dimethoxy-2-nitrobenzoic acid (3) was treated with thionylchloride and then reacted with ammonia to give4,5-dimethoxy-2-nitrobenzamide (4) as described by F. Nomoto et al.Chem. Pharm. Bull. 1990, 38, 1591-1595. The nitro group in compound (4)was reduced with sodium borohydride in the presence of copper sulfate(see C. L. Thomas Catalytic Processes and Proven Catalysts AcademicPress, New York (1970)) to give 4,5-dimethoxy-2-aminobenzamide (5) whichwas cyclized by refluxing with formic acid to give6,7-dimethoxyquinazoline-4(3H)-one (6). Compound (6) was refluxed withphosphorus oxytrichloride to provide the common synthetic precursor (7).

[0057] Compounds 1 and 2 (FIG. 4) were prepared from the commonsynthetic precursor (7) and the requsite aniline as follows.

[0058] 4-(4′-Hydroxylphenyl)-amino-6, 7-dimethoxyquinazoline (1). Amixture of 448 mg (2 mmol) of 4-chloro-6,7-dimethoxy-quinazoline (7) and2.5 mmol of 4-hydroxyaniline in 20 ml of alcohol (EtOH or MeOH) wasrefluxed for 8 hours. After cooling triethylamine was added to basifythe solution, and the solvent was concentrated to give material that wasrecrystallized from DMF to give compound (1); 84.29%; m.p. 245.0-248.0°C.; ¹H NMR (DMSO-d₆): δ 11.21(s, 1H, -NH), 9.70(s, 1H, -OH), 8.74(s, 1H,2-H), 8.22(s, 1H, 5-H), 7.40(d, 2H, J=8.9 Hz, 2′, 6′-H), 7.29(s, 1H,8-H), 6.85(d, 2H, J=8.9 Hz, 3′, 5′-H), 3.98(s, 3H, —OCH₃), 3.97(s, 3H,—OCH₃). UV(MeOH) λ_(max)(e): 203.0, 222.0, 251.0, 320.0 nm.IR(KBr)u_(max): 3428, 2836, 1635, 1516, 1443, 1234 cm⁻¹. GC/MS m/z 298(M⁺+1, 100.00), 297(M⁺, 26.56), 296(M⁺-1, 12.46).

[0059] 4-(3′-Bromo-4′-hydroxylphenyl)-amino-6,7-dimethoxy-quinazoline(2). A mixture of 448 mg (2 mmol) of 4-chloro-6,7-dimethoxy-quinazoline(7) and 2.5 mmol of 3-bromo-4-hydroxyaniline in 20 ml of alcohol (EtOHor MeOH) was refluxed for 8 hours. After cooling, triethylamine wasadded to basify the solution, and the solvent was concentrated to givematerial that was recrystallized from DMF to give compound (2); 89.90%;m.p. 233.0-233.5° C.; ¹H NMR(DMSO-d₆): δ 10.08(s, 1H, —NH), 9.38(s, 1H,-OH), 8.40(s, 1H, 2-H), 7.89(d, 1H, J_(2′, 5′)=2.7 Hz, 2′-H), 7.75(s,1H, 5-H), 7.55(dd, 1H, J_(5′, 6′)=9.0 Hz, J_(2′, 6′)=2.7 Hz, 6′-H),7.14(s, 1H, 8-H), 6.97(d, 1H, J_(5′, 6′)=9.0 Hz, 5′-H), 3.92(s, 3H,—OCH_(ER)), 3.90(s, 3H, —OCH₃). UV(MeOH)λ_(max)(e): 203.0, 222.0, 250.0,335.0 nm. IR(KBr)u_(max): 3431(br), 2841, 1624, 1498, 1423, 1244 cm⁻¹.GC/MS m/z 378(M⁺+2, 90.68), 377(M⁺1, 37.49), 376(M⁺, 100.00),360(M⁺3.63), 298(18.86), 282 (6.65).

Example 2 Biological Screening

[0060] Materials and Methods

[0061] Cell Lines. The establishment and characterization ofBTK-deficient, SYK-deficient, and LYN-deficient clones and reconstitutedSYK-deficient cell lines of DT-40 chicken lymphoma B-cells werepreviously reported. The culture medium was RPMI 1640 (LifeTechnologies; Gaithersburg, Md.), supplemented with 1% chicken serum(Sigma; St. Louis, Mo.), 5% fetal bovine serum (Hyclone, Logan, Utah)and 1% penicillin-streptomycin (Life Technologies). See Uckun, F. M.,Waddick, K. G., Mahajan, S., Jun, X., Takata, M., Bolen, J., andKurosaki, T. Science. 273: 1096-100, 1996; Kurosaki, T. Curr OpinImmunol. 9: 309-18, 1997; Kurosaki, T., Johnson, S. A., Pao, L., Sada,K., Yamamura, H., and Cambier, J. C. J. Exp. Med. 182: 1815-1823, 1995;and Dibirdik I., Kristupaitis D., Kurosaki T., Tuel-Ahlgren L., Chu A.,Pond D., Tuong D., Luben R., Uckun F. M. J. Biol. Chem. 273(7),pp:4035-4039, 1998.

[0062] Use of PTK Inhibitors. Cells (2×10⁶/ml) were treated for 24 hoursat 37° C. with either (1) the PTK inhibitory isoflavone genistein(Calbiochem, La Jolla, Calif.) at 111 mM (30 mg/ml) concentration or (2)the Janus family kinase, 3 (JAK-3)-specific PTK inhibitor4-(3′-bromo-4′-hydroxyphenyl)-amino-6,7-dimethoxyquinazoline,C₁₆,H₁₄Br(N₃O₃), kindly provided by Dr. Xing-Ping Liu, Alexander andParker Pharmaceutical Inc., Roseville, Minn.) at 270 MM (100 mg/ml)prior to radiation in order to assess the effects of these agents onradiation-induced c-jun activation.

[0063] Irradiation of cells. Cells (2×10⁶cells/ml) in plastic tissueculture flasks were irradiated with 10-20 Gy at a dose rate of 4 Gy/minduring log phase growth and under aerobic conditions using a ¹³⁷Csirradiator (J.L. Shephard, Glendale, Calif., as previously described byTuel Ahlgren, L., Jun, X., Waddick, K. G., Jin, J., Bolen, J., andUckun, F. M. “Role of tyrosine phosphorylation in radiation-induced cellcycle-arrest of leukemic B-cell precursors at the G2-M transitioncheckpoint,” Leuk Lymphoma. 20: 417-26, 1996; and Uckun, F. M., Jaszcz,W., Chandan Langlie, M., Waddick, K. G., Gajl Peczalska, K. and Song,C.W. “Intrinsic radiation resistance of primary clonogenic blasts fromchildren with newly diagnosed B-cell precursor acute lymphoblasticleukemia,” J Clin Inves. 91:1044-1051, 1993. In some experiments, cellswere preincubated with PTK inhibitors for 24 hours prior to irradiation.

[0064] c-jun probe. A 506 basepair (bp) c-jun probe was obtained bypolymerase chain reaction (PCR) amplification of chicken genomic DNA.Primer sequences were determined based upon the sequence of chickenc-jun (GenBank accession code CHKJUN). Two primers: 5′-ACTCTGCACCCAACTACAACGC-3′ (SEQ. ID NO: 1) and 5′-CTTCTACCGT CAGCTTTACGCG-3′ (SEQID NO: 2) were used for amplification. Amplification was performed witha mix of Taq polymerase and a proof reading polymerase (eLONGase:Taqpolymerase plus Pyrococcus species GB-D polymerase, Gibco BRL, GrandIsland, N.Y.) on an thermocycler, Ericomp Delta II cycler, using a hotstart. PCR products were subsequently cloned into the cloning vector,PCR 2.1 (Invitrogen, San Diego, Calif.). An insert of the proper size(506 basepair) was identified as chicken c-jun by sequence analysisusing PRISM dye terminator cycle sequencing (AmpliTaq® DNA Polymerase,FS) and analyzed on an automated sequencer, ALF express sequencer(Pharmacia Biotech, Piscataway, N.J.). A 538 base pair chickenglyceraldehyde 3-phosphate dehydrogenase (GAPDH) probe was generated byreverse transcription and subsequent PCR amplification (RT-PCR) fromchicken RNA with the following primers: 5′-AGAGGTGCTGCCCAGAACATCATC-3′(SEQ ID NO: 3) and 5′-GTGGGGAGACAGAAGGGAACAGA-3′ (SEQ ID NO: 4). A 413bp chicken B-actin probe was generated by RT-PCR amplification fromchicken RNA with the following primers: 5′-GCCCTCTTCCAGCATCTTTCTT-3′(SEQ ID NO: 5) and 5′-TTTATGCGCATTTATGGGTT-3′ (SEQ ID NO: 6). Theamplified cDNAs were cloned into PCR 2.1.

[0065] RNA isolation and Northern blot hybridization analysis. Total RNAwas extracted from approximately 2.5×10⁷ cells with Trizol Reagent, amonophasic solution of phenol and guanidine isothiocyanate as describedby Chomcznski, P. and Sacchi, N. “Single-step method of RNA isolation byguanidinium-thiocyanate-phenol-chloroform extraction,” Anal. Biochem.162: 156-159, 1987. Poly (A)⁺ RNA was isolated directly from 1-3×10⁸cells with an Invitrogen Fast Trak 2.0 mRNA isolation kit. In brief,cells were lysed in a sodium dodecyl sulfate (SDS) lysis buffercontaining a proprietary mixture of proteases. The lysate was directlyincubated with oligo-dT for absorption and subsequent elution of poly(A)⁺ RNA.

[0066] Two micrograms of poly (A)⁺ or 20 micrograms of total RNA weredenatured in formaldehyde/formamide loading dye at 65° prior to loadingonto a 1% agarose-formaldehyde denaturing gel. Transcript sizes weredetermined relative to RNA markers of 0.5-9 kb. The gels were stainedwith Radiant Red in H₂O to check loading and integrity of RNA prior totransfer. The RNA was subsequently transferred to positively chargednylon membrane with 20× standard sodium citrate(SSC) transfer buffer(LXSSC=0.15 M sodium chloride-0.015 M sodium citrate) by downwardcapillary transfer. The c-jun fragment was radiolabeled by randompriming with [(-³²P]-dCTP (3000 Ci/mM) [Amersham, Arlington Heights,Ill.] (40). Northern blots were hybridized overnight at 42° C. inprehybridization/hybridization solution (50% formamide with proprietaryblocking and background reduction reagents; Ambion, Austin, Tex.) for16-24 hours and unbound probe was removed by washing to a finalstringency of 0.1% SDS, 0.1×SSC (65° C.). The blots were analyzed bothby autoradiography and using the BioRad Storage Phosphor Imager System(BioRad, Hercules, Calif.) for quantitative scanning. The blots weresubsequently stripped in boiling 0.1% SDS, and then rehybridized with achicken GAPDH and/or chicken (62 -actin probe to normalize for loadingdifferences.

[0067] Results and Discussion

[0068] Exposure of DT40 chicken lymphoma B-cells to ionizing radiationactivates the c-jun protooncogene. Exposure of human lymphoma B-cells to10-20 Gy-rays results in enhanced c-jun expression with a maximumresponse at 1-2 hours (Chae, H. P., Jarvis, L. J., and Uckun, F. M.Cancer Res. 53: 447-51, 1993). It has also been reported that ionizingradiation triggers in DT-40 chicken lymphoma B-cells biochemical andbiological signals similar to those in human lymphoma B-cells (Uckun, F.M., Waddick, K. G., Mahajan, S., Jun, X., Takata, M., Bolen, J., andKurosaki, T. Science. 273: 1096-100, 1996). In order to determine ifDT-40 chicken lymphoma B-cells show a similar c-jun response to ionizingradiation, DT-40 cells were irradiated with 5,10,15 or 20 Gy andexamined total RNA harvested from cells 2 or 4 hours after radiationexposure for expression levels of 1.8 kb chicken c-jun transcripts byquantitative Northern blot analysis. As shown in FIG. 1A, radiationexposure increased the level of c-jun transcripts in a dose-andtime-dependent manner without significantly affecting the GAPDHtranscript levels with a maximum stimulation index (SI) [as determinedby comparison of the c-jun/GAPDH ratios in non-irradiated versusirradiated cells] of 3.1, 4 hours after 20 Gy. In seven additionalindependent experiments, the stimulation index for 20 Gy ionizingradiation at 2 hours after radiation exposure ranged from 2.4 to 3.8(mean (SE=2.9±0.4).

[0069] The role of PTK in radiation-induced activation of c-junexpression in chicken lymphoma B cells was examined next, since PTKinhibitors were shown to prevent radiation-induced c-jun activation inhuman lyrnphoma B-cells. As shown in FIG. 1B, ionizing radiation did notsignificantly enhance c-jun expression levels in DT-40 cells treatedwith the PTK-inhibitory isoflavone, genistein (stimulation index=1.1)indicating that activation of a PTK is required for radiation-inducedc-jun expression in chicken lymphoma B cells as well. These findingsestablished DT40 chicken lymphoma B-cells as a suitable model to furtherelucidate the molecular mechanism of radiation-induced c-jun activation.

[0070] Cytoplasmic protein tyrosine kinases BTK, LYN, and SYK are notrequired for radiation induced c-jun activation. BTK is abundantlyexpressed in lymphoma B-cells and its activation has been shown to berequired for radiation-induced apoptosis of DT-40 cells (Uckun, F. M.,Waddick, K. G., Mahajan, S., Jun, X., Takata, M., Bolen, J., andKurosaki, T. Science. 273: 1096-100, 1996). DT-40 cells renderedBTK-deficient by targeted disruption of the BTK genes do not undergoapoptosis after radiation exposure. Therefore, we set out to determineif BTK could be the PTK responsible for radiation-induced c-junactivation as well, by comparing the levels of c-jun induction inBTK-deficient (BTK) versus wild-type DT-40 cells. Contrary to ourexpectations, 20 Gy ionizing radiation did not fail to induce c-junexpression in BTK-deficient DT-40 cells in any of the three independentexperiments performed. The stimulation indices ranged from 1.6 to 3.9(mean±SE=2.4±0.5) (FIG. 2). Thus, ionizing radiation-induced increasesin c-jun transcript levels do not depend upon the presence of BTK.

[0071] Since SYK is also abundantly expressed in DT-40 cells and israpidly activated after ionizing radiation, we next examined if SYKmight be the PTK responsible for radiation-induced increases in c-juntranscript levels. As shown in FIG. 3A, 20 Gy ionizing radiationenhanced c-jun expression in SYK DT-40 cells rendered SYK-deficient bytargeted gene disruption even though the stimulation indices observed infive independent experiments were lower than from those in wild-typecells (1.9±0.2, vs 2.9±0.4,p<0.01). Thus, SYK is not required forradiation-induced c-jun activation in DT-40 cells but it may participatein generation of an optimal signal.

[0072] DT40 cells express high levels of LYN but do not express othermembers of the Src PTK family, including BLK, HCK, SRC, FYN, or YES atdetectable levels (see Uckun, F. M., Waddick, K. G., Mahajan, S., Jun,X., Takata, M., Bolen, J., and Kurosaki, T. Science. 273: 1096-100,1996; Kurosaki, T., Johnson, S. A., Pao, L., Sada, K., Yamamura, H., andCambier, J. C. “Role of the Syk autophosphorylation site and SH2 domainsin B cell antigen receptor signaling,” J. Exp. Med. 182: 1815-1823,1995; and Takata, M., Homma, Y., and Kurosaki, T. “Requirement ofphospholipase C-γ2 activation in surface immunoglobulin M-induced B cellapoptosis.,” J Exp Med. 182: 907-914, 1995. Since it has previously beendemonstrated that SRC family PTK are essential for UV-stimulatedincreases in c-jun expression, we postulated that the predominantSRC-family member, LYN, might mediate radiation-induced c-jun expressionin DT-40 cells. To test this hypothesis, we examined the ability ofionizing radiation to activate c-jun expression in DT-40 cells renderedLYN-deficient by targeted gene disruption. LYN-deficient (LYN) cellsshowed enhanced c-jun expression after irradiation, however thestimulation indices were lower than those in wild-type DT-40 (FIG. 3B).Since LYN and SYK have been shown to cooperate in the generation ofother signals in B-cells (see Kurosaki, T. “Molecular mechanisms in Bcell antigen receptor signaling,” Curr Opin Immunol. 9: 309-18, 1997),the ability of ionizing radiation to induce c-jun expression in LYN⁻SYK⁻DT-40 cells, generated by targeted disruption of the syk gene in LYN⁻deficient DT-40 cells was examined. As shown in FIG. 3B, LYN SYK DT-40cells showed elevated c-jun transcript levels after irradiation,indicating that the c-jun response does not depend on either of thesePTK, either alone or in cooperation. Similar to SYK, LYN is not requiredfor radiation-induced c-jun activation in DT-40 cells but it mayparticipate in generation of an optimal response.

[0073] Interestingly, in four independent experiments, we observedhigher baseline expression levels of c-jun in SYK⁻ DT-40 cells than inwild-type DT-40 cells (Range: 1.4-2.3-fold, mean±SE=1.6+0.2-fold),suggesting that Syk may be involved in regulation of baseline c-junlevels. To further explore this possibility, we compared c-jun levels inSYK⁻ cells to those of SYK⁻ cells reconstituted with wild-type or kinasedomain mutant (K⁻) syk gene. We observed that reconstitution withwild-type syk reduced the higher baseline expression levels of c-jun inSYK⁻ cells, whereas reconstitution with a K⁻ syk failed to reduce c-junlevels (data not shown). These results implicate SYK as a negativeregulator of c-jun expression. This novel function of SYK seems todepend on its kinase domain.

[0074] Effects of a JAK-3 inhibitor on radiation-induced c-junactivation in DT40-cells. B-cell signal transduction events directfundamental decisions regarding cell survival during periods ofoxidative stress. A better understanding of the dynamic interplaybetween B-cell signaling pathways is needed to determine how vitaldecisions are dictated during intracellular oxidation changes. STATproteins (signal transducers and activators of transcription) are afamily of DNA binding proteins that were identified during a search forinterferon (IFN) a- or g-stimulated gene transcription targets. Thereare presently seven STAT family members. The JAK family of cytoplasmicprotein kinases were originally demonstrated to also function in IFNsignaling, and are now known to participate in a broad range ofreceptor-activated signal cascades. Different ligands and cellactivators employ specific JAK and STAT family members. The basic modelfor STAT activation suggests that in unstimulated cells, latent forms ofSTATs are predominantly localized within the cytoplasm. Ligand bindinginduces STAT proteins to associate with intracellular phosphotyrosineresidues of transmembrane receptors. Once STATs are bound to receptors,receptor-associated JAK kinases phosphorylate the STAT proteins. STATproteins then dimerize through specific reciprocal SH2-phosphotyrosineinteractions and may form complexes with other DNA-binding proteins.STAT complexes translocate to the nucleus and interact with DNA responseelements to enhance transcription of target genes. Signaling eventsregulating apoptotic responses have been shown to utilize STAT proteins.Notably, a recent study demonstrated JAK activation by tyrosinephosphorylation in cells that are exposed to reactive oxygenintermediates, which in-turn lead to tyrosine phosphorylation andactivation of STAT-1, STAT-3 and STAT-6.

[0075] After establishing that LYN, BTK, and SYK kinases are notrequired for radiation-induced c-jun activation, we set out to determineif c-jun activation is functionally linked to the JAK-STAT pathway. Tothis end, we examined the effects of a JAK-3 inhibitory novelquinazoline derivative on c-jun expression levels in irradiated DT-40cells. To identify a potent JAK-3 specific inhibitor, the effects of twonovel quinazoline derivatives on the enzymatic activity of JAK-1; JAK-2,and JAK-3 were examined using Sf21 cells that were infected withbaculovirus expression vectors for these kinases, using standard methods(FIG. 4). Infected cells were harvested, JAKs were immunoprecipitatedwith appropriate antibodies (anti-JAK-1: (HR-785), cat# sc-277, rabbitpolyclonal IgG affinity purified, 0.1 mg/ml, Santa Cruz Biotechnology;anti-JAK-2: (C-20)-G, cat # sc-294-G, goat polyclonal IgG affinitypurified, 0.2 mg/ml, Santa Cruz Biotechnology; anti-JAK-3: (C-21), cat #sc-513, rabbit polyclonal IgG affinity purified, 0.2 mg/ml, Santa CruzBiotechnology), and kinase assays were performed following a 1 hourexposure of the immunoprecipitated Jaks to the quinazoline compounds, asdescribed by Uckun, F. M., Waddick, K. G., Mahajan, S., Jun, X., Takata,M., Bolen, J., and Kurosaki, T. Science. 273: 1096-100, 1996; Uckun F.M, Evans W. E, Forsyth C. J, Waddick K. G, T-Ahlgren L., Chelstrom L. M,Burkhardt A., Bolen J., Myers D. E. Science 267:886-891, 1995; and MyersD. E., Jun X., Waddick K. G., Forsyth C., Chelstrom L. M., Gunther R.L., Turner N. E, Bolen J., Uckun F. M. Proc Nat'l Acad Sci USA 92:9575-9579, 1995; and Tuel Ahlgren, L., Jun, X., Waddick, K. G., Jin, J.,Bolen, J., and Uckun, F. M. Leuk Lymphoma. 20: 417-26, 1996.

[0076] As shown in FIG. 4B, both compounds inhibited JAK-3 (FIGS. B.3and B.4) but not JAK-1 (FIG. B.1) or JAK-2 (FIG. B.2) (FIG. 4D).Electrophoretic Mobility Shift Assays (EMSAs) were performed to examinethe effects of both compounds on cytokine-induced STAT activation.Specifically, 32Dc11/IL2Rβ cells (gift from James Ihle, St. JudeChildren's Research Hospital) were exposed at 8×10⁶/ml in RPMIsupplemented with FBS to the JAK-3 inhibitors at a final concentrationof 10 μg/ml in 1% DMSO) for 1 hour and subsequently stimulated with IL2or IL3 as indicated. Cells were collected after 15 minutes andresuspended in lysis buffer (100 mM Tris-HCI pH 8.0, 0.5% NP-40, 10%glycerol, 100 mM EDTA, 0.1 mM NaVO3, 50 mM NaF, 150 mM Nacl, 1 mM DTT, 3(g/ml Aprotinin, 2 g/ml Pepstatin A, 1 (g/ml Leupeptin and 0.2 mM PMSF).Lysates were precleared by centrifugation for 30 minutes. Cell extracts(approximately 10 g) were incubated with 2 μg of poly(dI-dC) for 30minutes, followed by a 30 minute incubation with 1 ng of poly nucleotidekinase-”³²P labeled double stranded DNA oligonucleotide representing theIRF-1 STAT DNA binding sequence (Santa Cruz Biotechnology, Santa Cruz,Calif.). Samples were resolved by nondenaturing PAGE and visualized byautoradiography. As shown in FIG. 4C, both compounds inhibited theJAK-3-dependent STAT activation after stimulation with IL-2, but theydid not affect the JAK-1/JAK-2-dependent STAT activation afterstimulation with IL-3. Compound 2 was selected for further experimentsdesigned to examine the effects of JAK-3 inhibition on radiation-inducedc-jun activation.

[0077] As shown in FIG. 5, ionizing radiation failed to induce c-junexpression in DT-40 cells treated with the JAK-3 inhibitor. Thisdemonstrates that JAK-3 inhibitors are capable of inhibiting radiationinduced c-jun expression.

[0078] In untreated cells, c-jun expression is induced by exposure toDNA-damaging chemical agents and by exposure to radiation. Thus, c-junexpression is an early marker of cellular response to such DNA-damagingagents. It has been shown that compounds that inhibit JAK-3 are capableof inhibiting the expression of c-jun. Accordingly, JAK-3 inhibitors maybe useful to prevent or treat diseases or conditions that result fromexposure to DNA-damaging agents.

[0079] JAK-3 maps to human chromosome 19p12-13.1. A cluster of genesencoding protooncogenes and transcription factors is also located nearthis region. JAK-3 expression has been demonstrated in mature B-cells aswell as B-cell precursors. JAK-3 has also been detected in leukemicB-cell precursors and lymphoma B-cells. The physiological roles forJAK-3 have been borne out through targeted gene disruption studies inmice, the genetic analysis of patients with severe combinedimmunodeficiency, and biochemical studies of JAK-3 in cell lines. A widerange of stimuli result in JAK-3 activation in B-cells, includinginterleukin 7 and interleukin 4. The B-cell marker CD40 constitutivelyassociates with JAK-3 and ligation of CD40 results in JAK-3 activationwhich has been shown to be mandatory for CD40-mediated gene expression.Constitutive activity of JAK-3 has been observed in v-abl transformedpre-B cells and coimmunoprecipitations show that v-abl physicallyassociates with JAK-3 implicating JAK-3 in v-abl induced cellulartransformation. See Ihle, J. N. “Janus kinases in cytokine signalling,”Philos Trans R Soc Lond B Biol Sci 351:159-66, 1996; Leonard, W. J.“STATs and cytokine specificity,” Nat Med 2:968-9, 1996; Levy, D. E.“The house that Jak/Stat built,” Cytokine Growth Factor Rev 8:81-90,1997; Riedy, M. C. et al. “Genomic sequence, organization, andchromosomal localization of human JAK-3,” Genomics 37, 57-61, 1996;Safford, M. G., Levenstein, M., Tsifrina, E., Amin, S., Hawkins, A. L.,Griffin, C.A., Civin, C.I. and Small, D. “JAK-3: expression and mappingto chromosome 19p12-13.1” [published erratum appears in Exp Hematol 1997July; 25(7):650]. Exp Hematol 25, 374-86, 1997; Kumar, A., Toscani, A.,Rane, S. and Reddy, E. P. “Structural organization and chromosomalmapping of JAK-3 locus,” Oncogene 13, 2009-14, 1996; Hoffman, S. M.,Lai, K. S., Tomfohrde, J., Bowcock, A., Gordon, L. A. and Mohrenweiser,H. W. “JAK-3 maps to human chromosome 19p12 within a cluster ofproto-oncogenes and transcription factors,” Genomics 43, 109-111, 1997;Tortolani, P. J. et al. “Regulation of JAK-3 expression and activationin human B cells and B cell malignancies,” J Immunol 155, 5220-6, 1995;Sharfe, N., Dadi, H. K., J J, O. S. and Roifnan, C. M. “JAK-3 activationin human lymphocyte precursor cells,” Clin Exp Immunol 108, 552-6, 1997;Gurniak, C. B. and Berg, L. J. “Murine JAK-3 is preferentially expressedin hematopoietic tissues and lymphocyte precursor cells,” Blood 87,3151-60, 1996; Rolling, C., Treton, D., Beckmann, P., Galanaud, P. andRichard, Y. “JAK-3 associates with the human interleukin 4 receptor andis tyrosine phosphorylated following receptor triggering,” Oncogene 10,1757-61, 1995; Rolling, C., Treton, D., Pellegrini, S., Galanaud, P. andRichard, Y. “IL4 and IL13 receptors share the gamma c chain and activateSTAT6, STAT3 and STAT5 proteins in normal human B cells,” FEBSLett 393,53-6, 1996; Hanissian, S. H. and Geha, R. S. “JAK-3 is associated withCD40 and is critical for CD40 induction of gene expression in B cells,”Immunity 6, 379-87, 1997; Danial, N. N., Pernis, A. and Rothman, P. B.“Jak-STAT signaling induced by the v-abl oncogene,” Science 269, 1875-7,1995.

[0080] Summary

[0081] Exposure of B-lineage lymphoid cells to ionizing radiationinduces an elevation of c-jun protooncogene mRNA levels. This signal isabrogated by protein tyrosine kinase (PTK) inhibitors, indicating thatactivation of an as yet unidentified PTK is mandatory forradiation-induced c-jun expression. Experimental evidence shows that thecytoplasmic tyrosine kinases BTK, SYK and LYN are not required for thissignal. Lymphoma B-cells rendered deficient for LYN, SYK or both bytargeted gene disruption showed increased c-jun expression levels afterradiation exposure, but the magnitude of the stimulation was lower thanin wild-type cells. Thus, these PTK may participate in the generation ofan optimal signal. Notably, inhibitors of Janus family kinase 3 (JAK-3)abrogated radiation-induced c-jun activation. This suggests that JAKsare important regulators of radiation-induced c-jun activation, and thatJAK-3 inhibitors are useful for preventing or treating diseases orconditions that result from chemical-induced or radiation-induced c-junactivation.

[0082] All publications, patents, and patent documents are incorporatedby reference herein, as though individually incorporated by reference.The invention has been described with reference to various specific andpreferred embodiments and techniques. However, it should be understoodthat many variations and modifications may be made while remainingwithin the spirit and scope of the invention.

What is claimed is:
 1. A method comprising inhibiting c-jun activationin mammalian or avian cells by contacting the cells with a substancethat inhibits the activity of Janus family kinase 3 (JAK-3).
 2. Themethod of claim 1 wherein the c-jun activation results from exposure ofthe cells to ara-C, a topoisomerase II inhibitor, ultraviolet radiation,an alkylating agent, or ionizing radiation.
 3. The method of claim 1wherein the c-jun activation results from exposure of the cells toultraviolet radiation or ionizing radiation.
 4. The method of claim 1wherein the contacting is performed in vitro.
 5. The method of claim 1wherein the contacting is performed in vitro.
 6. The method of claim 2wherein the contacting occurs prior to the exposure.
 7. The method ofclaim 2 wherein the contacting occurs after the exposure.
 8. The methodof claim 1 wherein the substance is a protein.
 9. The method of claim 1wherein the substance is a compound of formula I:

wherein X is HN, R₁₁N, S, O, CH₂, or R₁₁CH; R₁₁ is hydrogen,(C₁-C₄)alkyl, or (C₁-C₄)alkanoyl; R₁-R₈ are each independently hydrogen,hydroxy, mercapto, amino, nitro, (C₁-C₄)alkyl, (C₁-C₄)alkoxy,(C₁-C₄)alkylthio, or halo; wherein two adjacent groups of R₁-R₅ togetherwith the phenyl ring to which they are attached may optionally form afused ring, for example forming a naphthyl or a tetrahydronaphthyl ring;and further wherein the ring formed by the two adjacent groups of R₁-R₅may optionally be substituted by 1, 2, 3, or 4 hydroxy, mercapto, amino,nitro, (C₁-C₄)alkyl, (C₁-C₄)alkoxy, (C₁-C₄)alkylthio, or halo; and R₉and R₁₀ are each independently hydrogen, (C₁-C₄)alkyl, (C₁-C₄)alkoxy,halo, or (C₁-C₄)alkanoyl; or R₉ and R₁₀ together are methylenedioxy; ora pharmaceutically acceptable salt thereof.
 10. The method of claim 1wherein the substance is4-(4′-hydroxylphenyl)-amino-6,7-dimethoxyquinazoline or4-(3′-bromo-4′-hydroxylphenyl)-amino-6,7-dimethoxyquinazoline; or apharmaceutically acceptable salt thereof.
 11. The method of claim 1wherein the cells are mammalian.
 12. The method of claim 1 wherein thecells are human.
 13. The method of claim 1 wherein the cells are avian.14. A therapeutic method for preventing or treating a pathologicalcondition in a mammal wherein c-jun activation is implicated andinhibition of its activation is desired comprising administering to amammal in need of such therapy, an effective amount of a substance thatinhibits the activity of JAK-3.